Hi proteo.
I'm not quite sure I understand what your problem is! From what I understand, you try to separate 2 proteins but they are too similar in mass. Is that it?
If so you could do a step of IEF (isoelectrical focusing) prior to SDS-PAGE. 2D-PAGE should get you there.

As your sample shows sinlge band on SDS PAGE and two on native PAGE, it indicates thatthey have similar mol.wt but slightly dissimmilar charge. Did you inlcude ion exchange chromatography in you protocol. Hydroxylapatite chromatography generallt resolves such close bands. Chramtofocussing can be used as separation is based on charge.

Thank sharath and Simon
Now i try to use Mono Q column for separating two protein, but my enzyme still bound to the column, even used 4 M NaCl in 50 mM Tris-HCl, pH 8.0 + 1 mM CaCl2. If you have some recommentation, please tell me.